The molecular pathogenesis of fully virulent, wild-type Y. pestis in relevant animal models has been relatively neglected because of the scarcity of secure BSL-3 facilities and trained personnel certified to work with this Class A select agent. The threat of bioterrorism and the emergence of multiply-antibiotic resistant strains of Y. pestis increases the urgency for a more detailed understanding of the host-pathogen relationship at the molecular level that may lead to the design of improved medical countermeasures and diagnostics. We have established mouse and rat models of bubonic plague that incorporate flea-to-rodent transmission to investigate the role of specific Y. pestis virulence factors and to characterize the host response to naturally acquired infection. We have characterized the kinetics, microbiology, and histopathology of bubonic plague in rats following intradermal injection of Y. pestis, and used this model to characterize the gene expression profile of Yersinia pestis in the infected lymph node during bubonic plague using whole-genome microarray technology. Our previous work has shown that three important Y. pestis virulence factors, Ail (a Y. pestis outer surface protein), the Type III secretion system (T3SS) encoded on the Yersinia virulence plasmid, and the plasminogen activator (Pla) encoded on the 9.5-kb Y. pestis-specific plasmid all act to limit the polymorphonuclear leukocyte response to bubonic plague infection in vivo (polymorphonuclear leukocytes, also referred to as PMNs or neutrophils, are phagocytic cells that are an important innate defense against infection). Thus, we now have several lines of evidence that the PMN response correlates with successful outcome to infection, and this aspect of host-pathogen interaction has become a focus of our lab. We discovered that the Y. pestis T3SS effector protein YopJ strongly inhibits the secretion of the IL-8 by human neutrophils (submitted for publication during fiscal year 2015). In addition, we reported that a small but significant percentage of Y. pestis, even T3SS-negative attenuated strains, survive and eventually replicate within phagosomes after being ingested by neutrophils. Infected neutrophils were also demonstrated to be taken up by macrophages in vitro, where Y. pestis could continue to replicate. The results indicate that neutrophil phagocytosis is not invariably fatal to Y. pestis, and that virulence factors other than the T3SS that counteract the strongly bactericidal environment of the neutrophil phagosome are important to plague pathogenesis. This has been the focus of research of a post-baccalaureate fellow during the current year During the past year, we expanded our intravital microscopy capabilities to include imaging the skin-draining lymph node (dLN) early after infection with Y. pestis. We observed Y. pestis in the dLN within minutes after intradermal injection and examined potential interactions between the bacteria, subcapsular sinus macrophages and neutrophils. The majority of the rapidly disseminating bacteria appear to be non-cell-associated, remain confined to the subcapsular sinus of the dLN and interact minimally with neutrophils and subcapsular sinus macrophages. Passive immunization of animals with anti-Y. pestis antibody (Ab) is known to provide complete protection in models of bubonic plague. The precise mechanisms responsible for the antibody-mediated immunity to Y. pestis remain unknown. Building upon our previous studies examining the neutrophil response to intradermal Y. pestis in nave animals, we began investigating the effects of antibody opsonization on these responses in the skin and dLN after infection. We found that the presence of anti-Y. pestis antibody dramatically increases interactions between neutrophils and Y. pestis in the dermis and dLN and appears to lead to rapid clearance of bacteria from the dermis. We, and others, have reported that while the majority of Y. pestis taken up by neutrophils are quickly killed, approximately 10-15% of the bacteria survive and eventually replicate intracellularly. We investigated the mechanism(s) employed by Y. pestis to survive the highly microbicidal environment within a neutrophil. We tested several mutant strains of Y. pestis that have been deleted of known virulence factors, including the attachment and invasion locus protein (ail), the transcription factors PhoP/Q and OxyR, and the F1 protein capsule. None of the factors affected survival of Y. pestis within PMNs, but the F1 capsule mutant was phagocytosed much more readily than WT. We have also established immunofluorescence microscopy techniques that allow differentiation of live vs. dead bacteria and characterization of Y. pestis-containing vacuoles within neutrophils. To facilitate our studies involving in vitro assays of Y. pestis-neutrophil interactions we have established a system in the lab for the generation of immortalized murine neutrophil progenitor cells based on retroviral transduction of a Hoxb8-estrogen receptor construct in to bone marrow cells described by Wang et al. (ref: Nat Methods. 2006 Apr;3(4):287-93). We have successfully used this method to generate large numbers of murine neutrophils suitable for a variety of in vitro assays. We are now using a second in vivo imaging system (IVIS) in conjunction with bioluminescent Y. pestis strains to monitor the incidence and dissemination patterns of infection in mice challenged by flea bite. Fleas will be used within the first week after infection to evaluate what is known as early-phase transmission, and again 2-3 weeks after infection to evaluate a second, distinct transmission mechanism that is dependent on Y. pestis biofilm formation in the flea foregut. Results will be compared to previous studies that used intradermal inoculation by needle. In 2016, we also evaluated the cytokine response of human PMNs following incubation with the pathogenic Yersinia species. Y. pestis strains with the pCD1 virulence plasmid, which encodes cytotoxic Yop proteins that are translocated into host cells, stimulated little or no cytokine production compared to pCD1-negative strains. In particular, PMNs incubated with pCD1-negative Y. pestis secreted 1,000-fold higher levels of interleukin-8 (IL-8 or CXCL8), a proinflammatory chemokine important for PMN recruitment and activation. Deletion of yopE, -H, -T, -M or ypkA had no effect on pCD1-dependent inhibition, whereas deletion of yopJ resulted in significantly increased IL-8 production. Like Y. pestis, the enteropathogenic Yersinia species inhibited IL-8 secretion by PMNs, and strains lacking the virulence plasmid induced high levels of IL-8. Our results show that virulence plasmid-encoded effector Yops, particularly YopJ, prevent IL-8 secretion by human PMNs. Suppression of the chemotactic IL-8 response by Y. pestis may contribute to the delayed PMN recruitment to the infected lymph node that typifies bubonic plague.